A pearl fusion protein with whitening effects, its preparation method and application

Abstract

This invention belongs to the field of synthetic biology, specifically relating to a pearl fusion protein with whitening effects, its preparation method, and its applications. The fusion protein of this invention is derived from the matrix protein of the *Hylocereus undatus* shell, and its amino acid sequence is shown in SEQ ID No:2. The results of the examples show that the fusion protein has high purity, is non-toxic to cells, and can significantly downregulate the expression level of the h-TYR gene in human melanoma cells SK-MEL-28. At a concentration of 1 mg / mL, it can inhibit tyrosinase activity by 87.6%, exhibiting significant whitening effects. It also possesses antioxidant and moisturizing functions, significantly increasing the moisture content of the stratum corneum. Human skin occlusive patch tests have verified that it is safe and non-irritating to human skin and can be widely used in cosmetics, medical aesthetic products, and other fields.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a pearl fusion protein with whitening effects, its preparation method, and its application. Background Technology

[0002] The triangular sail mussel (Hypriosis cumingii) is a freshwater pearl-producing mussel endemic to China, accounting for 80% of the total freshwater pearl production and holding a very important position in the pearl industry. Both the shell and pearl of the triangular sail mussel are products of calcium carbonate biomineralization. The biomineralization process leading to calcium carbonate crystals is guided and regulated by a series of matrix proteins. Zeng Shimei et al. identified two new shell matrix protein genes, hic31 and hic52, from the mantle of the triangular sail mussel and conducted preliminary studies on their structure and function (Zeng Shimei. Identification and Biomineralization Function Study of Triangular Sail Mussel Shell Matrix Protein Genes hic31 and hic52 [D]. Shanghai Ocean University [2026-06-11]. DOI:CNKI:CDMD:2.1016.912275.).

[0003] The full-length cDNA sequence of the hic31 gene is 1432 bp, encoding 318 amino acids, of which amino acids 1-18 form a signal peptide. After removing the signal peptide, the theoretical molecular weight is 28.82 kDa, with an isoelectric point of 7.00, indicating a neutral matrix protein. The amino acid sequence of the hic31 protein is rich in glycine (Gly), accounting for 26.67% of the total amino acids. Gly often clusters at the N-terminus, forming four glycine clusters in the form of (Gly)n (n=3-7). Met / Ser often accompanies these glycine clusters, forming (Gly)mX(Gly)n (m>1, n>1, X refers to Met / Ser) repeats in the sequence. The secondary structure of the hic31 protein is mainly α-helical, and its higher-order structure is predicted to be a filamentous protein, showing structural similarity to type I collagen (α1 and α2). Based on comprehensive analysis, the hic31 protein is a prism-layer matrix protein, likely participating in prism mineralization as a framework protein.

[0004] The full-length cDNA sequence of the hic52 gene is 2053 bp, encoding 543 amino acids, of which the signal peptide is the first 18 amino acids. Excluding the signal peptide, the predicted theoretical molecular weight is 52.2 kDa, and the isoelectric point is 10.37, indicating it is a basic protein. Amino acid composition analysis revealed that the hic52 protein is composed of a large amount of Gly (28.8%) and Gln (12.4%), exhibiting a high frequency of repetitive fragments in the sequence: (1) Gly mainly forms multiple glycine clusters (Gly)n (n>2) at the N-terminus; (2) Gln and Asn are mainly distributed in the middle and C-terminus of the sequence, and there are two special repetitive regions, namely (Gln)2Asn(Gln)4(Asn)2 and (Gln)2Asn(Gln)4(Asn)2. )4Asn(Gln)4(Asn)2; (3) There are two consecutive Thr-Ser-Pro, two symmetrical consecutive Thr-Asn-Gln repeats, and two Gly-Gln-X-Gly-Asn-Gly-Gln repeats (X refers to Gln and Glu) in the middle of the sequence; (4) At the C-terminus, there is a (Gly-X-Gly-Y)n repeat, where X and Y are M / S / Q / G / F / I / N. All these motifs are very likely related to calcium binding or crystal interaction. Secondary structure prediction revealed that hic52 protein contains a long α-helix structure composed of 29 amino acids at the C-terminus, and the higher-order structure of hic52 protein also shows a certain degree of structural similarity with type I collagen (α1 and α2). Comprehensive analysis shows that hic52 protein is mainly a matrix protein related to nacre formation.

[0005] Currently, most pearl-based whitening products on the market are used in whole-powder form. It is generally believed that the finer the pearl powder, the higher the absorption rate and the better the effect. However, the active whitening proteins in pearl are not yet fully understood, leading to indiscriminate use of pearl and low utilization efficiency of active ingredients. Therefore, the technical problem this invention aims to solve is: to provide a protein with clearly defined whitening effects. Summary of the Invention

[0006] The purpose of this invention is to provide a pearl fusion protein with whitening effect, its preparation method and application. The fusion protein provided by this invention has a significant tyrosinase inhibition whitening effect, while also having antioxidant and moisturizing functions, and has good biosafety and is non-irritating to human skin.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a pearl fusion protein with whitening effects: The amino acid sequence of the fusion protein is shown in SEQ ID No:2; Alternatively, the fusion protein may be a derived protein that has one or more amino acids substituted, deleted, or added to the amino acid sequence shown in SEQ ID No:2, and has the activity of the protein shown in SEQ ID No:2.

[0008] The present invention also provides a gene encoding the above-mentioned fusion protein.

[0009] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID No:1.

[0010] The present invention also provides a recombinant plasmid comprising the above-mentioned gene.

[0011] The present invention also provides an engineered bacterium, wherein the engineered bacterium comprises the above-mentioned recombinant plasmid.

[0012] This invention also provides a method for preparing a pearl fusion protein with whitening effects, comprising the following steps: S1. Based on the functional region sequences of HIC31 and HIC52, the matrix proteins of the Triangular Sail Mussel, the properties of the pET28a(+) vector, and the host codon adaptability of Escherichia coli, the gene sequence and amino acid sequence of the fusion protein were designed and obtained using bioinformatics analysis software, and then the gene was synthesized. S2, based on the gene sequence of the fusion protein obtained in S1, construct the pET28a(+)-HIC31 / HIC52 vector; S3, the pET28a(+)-HIC31 / HIC52 vector was transformed into Escherichia coli competent cells to obtain engineered bacteria BL21-pET28a(+)-HIC31 / HIC52; S4. After inducing expression of the engineered bacteria BL21-pET28a(+)-HIC31 / HIC52, the bacterial cell precipitate was collected by centrifugation, the supernatant was obtained by lysis, and the fusion protein was obtained by purification.

[0013] Preferably, the method further includes S5: freeze-drying the fusion protein obtained in S4.

[0014] Preferably, step S2 of the method specifically includes the following steps: S21, the plasmid pET28a(+) and the plasmid PUC57-HIC31 / HIC52 were double-digested with NcoI and HindIII, respectively; S22. After the reaction was completed, the enzyme digestion system was subjected to electrophoresis on an agarose gel, and the double enzyme digestion products pET28a(+)(NcoI+HindIII) and HIC31 / HIC52(NcoI+HindIII) were recovered using a gel recovery kit. S23, the recovered double digestion product is ligated using Solution I ligase at 16°C for 0.5-3 hours; each 10 μL ligation system includes: 1 μL vector fragment, 4 μL target gene, and 5 μL Solution I ligase; S24. The recombinant plasmid system ligated from S23 was transformed into E. coli DH5α competent cells. After LB (Kana) resistance selection, positive transformants were selected and cultured. After PCR identification, sequencing was performed. If the sequencing results were correct, the pET28a(+)-HIC31 / HIC52 vector was successfully constructed. The pET28a(+)-HIC31 / HIC52 vector was extracted using a plasmid miniprep kit.

[0015] Preferably, when performing double enzyme digestion, each 50ul enzyme digestion system includes: 4ul of FastDigest NcoI, 4ul of FastDigest HindIII, 5ul of 10×FastDigest Green Buffer, 12ul of ddH2O, and 25ul of pET28a(+) or PUC57-HIC31 / HIC52; More preferably, the enzyme digestion conditions are: digestion at 37°C for 20 min in a PCR instrument.

[0016] Preferably, step S3 of the method specifically includes the following steps: Mix 1 μL of pET28a(+)-HIC31 / HIC52 plasmid with thawed E. coli BL21(DE3) competent cells and incubate on ice for 30 min. Heat shock in a 42°C water bath for 90 s, then incubate on ice again for 5 min. Add 400 μL of antibiotic-free LB medium and incubate on a shaker at 37°C and 190 rpm for 40 min. Spread 200 μL of the bacterial culture evenly onto an LB agar plate containing 50 μg / ml kanamycin (Kana). Incubate upside down at 37°C overnight. The next day, pick transformed colonies for inoculation and culture. Extract the plasmid from the overnight culture of transformed colonies. If the nucleotide sequencing is correct, the recombinant fusion protein engineered bacteria BL21-pET28a(+)-HIC31 / HIC52 is obtained.

[0017] Preferably, step S4 of the method specifically includes the following steps: S41, pick a single colony of BL21-pET28a(+)-HIC31 / HIC52, inoculate it into 5 ml of LB (Kana) medium, and incubate overnight at 37°C with shaking at 180 rpm; S42, take 300ul of overnight bacterial culture at a 1% inoculation rate and inoculate it into an Erlenmeyer flask containing 30ml of LB (Kana) medium. Incubate for 2-3 hours until the initial OD600 reaches 0.6-0.8, and then perform IPTG induction. S43, the final concentration of the inducer IPTG can be selected from 0.2~1mM, the induction temperature can be selected from 16~37℃, and the induction time can be selected from 5~12h; After S44 induction, the bacterial cell precipitate was collected by centrifugation, the supernatant was obtained by disruption, and the fusion protein stock solution was purified.

[0018] Further, preferably, step S44 specifically includes the following steps: S441 resuspended the induced BL21-pET28a(+)-HIC31 / HIC52 cells thoroughly in a 10× solution of 20mM PB + 1M sodium chloride (pH 5.8), ruptured the cells using an autoclave, centrifuged, collected the supernatant, added 20mM PB (pH 5.8) to the supernatant, adjusted the conductivity, and filtered through a 0.45μm membrane.

[0019] S442 purifies the filtered filtrate by chromatography, then washes it through the column with solution A (20 mM PB, 0.4 M sodium chloride (pH 5.8)) to remove unbound proteins until the UV baseline is stable. S443 is then passed through a chromatography column with solution B (20 mM PB, 1.5 M sodium chloride (pH 5.8) and eluted with 100% B / 10 CV. The protein corresponding to the elution peak is collected to obtain the HIC31 / HIC52 fusion protein stock solution.

[0020] Preferably, step S5 specifically includes the following steps: freeze-drying the fusion protein stock solution using a vacuum freeze dryer to prepare freeze-dried powder.

[0021] This invention also provides the application of the pearl fusion protein with whitening effects as described above, the application including any of the following: Applications in reducing the expression level of tyrosinase genes or inhibiting tyrosinase activity; In ABTS + Applications in free radical scavenging or antioxidation; Applications in skin whitening or maintaining the moisture content of the stratum corneum; Applications in the preparation of cosmetics or medical aesthetic products.

[0022] Beneficial effects: 1. The fusion protein described in this invention has no obvious toxicity to cells and good biosafety, laying a safe foundation for its application.

[0023] 2. The fusion protein of the present invention can significantly downregulate the expression level of the h-TYR gene in human melanoma cells SK-MEL-28, indicating that the fusion protein has a whitening effect by inhibiting melanin synthesis.

[0024] 3. The fusion protein described in this invention can inhibit tyrosinase activity by up to 87.6% at a concentration of 1 mg / mL, exhibiting a strong inhibitory effect on tyrosinase and significant whitening efficacy.

[0025] 4. The fusion protein described in this invention has certain antioxidant effects, which can effectively scavenge free radicals and delay skin oxidative damage.

[0026] 5. The fusion protein described in this invention was verified by a closed patch test on human skin. Among 30 subjects, 0 cases of adverse skin reactions occurred, indicating that it has good skin irritation safety.

[0027] 6. The fusion protein described in this invention can significantly increase the moisture content of the stratum corneum of the skin and has a certain moisturizing effect. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is an SDS-PAGE electrophoresis image of the bacterial cell lysate induced by BL21-pET28a(+)-HIC31 / HIC52 involved in this invention.

[0030] Figure 2 This is an SDS-PAGE electrophoresis image of the HIC31 / HIC52 fusion protein purification experiment involved in this invention.

[0031] Figure 3 This is a bar graph showing the cytotoxicity test results of the HIC31 / HIC52 fusion protein involved in this invention.

[0032] Figure 4 This is a bar chart showing the gene detection results of the HIC31 / HIC52 fusion protein involved in this invention.

[0033] Figure 5 This is a standard curve of ABTS+ free radical scavenging rate of glutathione.

[0034] Figure 6This is a standard curve of the ABTS+ free radical scavenging rate of the HIC31 / HIC52 fusion protein involved in this invention.

[0035] Figure 7 The bar chart shows the results of skin stratum corneum moisture content of the HIC31 / HIC52 fusion protein involved in this invention. Detailed Implementation

[0036] This invention provides a pearl fusion protein with whitening effects, its preparation method, and its application.

[0037] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0038] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0039] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0040] Example 1: Construction, identification and preparation of engineered bacteria containing BL21-pET28a(+)-HIC31 / HIC52 fusion protein 1.1 Design of target gene sequence Based on the functional region sequences of HIC31 and HIC52, the properties of the pET28a(+) vector, and the codon adaptability of Escherichia coli host, the gene sequence of the fusion protein was designed as shown in SEQ ID No:1.

[0041] It should be noted that in the gene sequence shown in SEQ ID No:1, the first 6 bases are NcoI restriction sites, the last 6 bases are HindIII restriction sites, and TAA is a stop codon.

[0042] The amino acid sequence of the fusion protein is shown in SEQ ID No:2.

[0043] It should be noted that in the amino acid sequence shown in SEQ ID No:2, the first 127 amino acids are the HIC31 protein domain and the last 171 amino acids are the HIC52 protein domain.

[0044] 1.2 Carrier Construction The gene sequence of the fusion protein (HIC31 / HIC52 protein) shown in SEQ ID No:1 was sent to Suzhou Genewise Biotech Co., Ltd. for gene synthesis, and detection primers were synthesized simultaneously. T7: SEQ ID No:3 T7ter: SEQ ID No:4 Plasmid pET28a(+) and plasmid PUC57-HIC31 / HIC52 synthesized by Suzhou Genewiz Biotechnology Co., Ltd. were double-digested with NcoI and HindIII, respectively. The digestion system (50 μL) consisted of: FastDigest NcoI 4 μL, FastDigest HindIII 4 μL, 10×FastDigest Green Buffer 5 μL, ddH2O 12 μL, and pET28a(+) or PUC57-HIC31 / HIC52 25 μL. Digestion was performed at 37°C for 20 min in a PCR instrument. After the reaction, electrophoresis was performed on a 1% agarose gel, and the products of pET28a(+)(NcoI+HindIII) and HIC31 / HIC52(NcoI+HindIII) were recovered using a gel extraction kit.

[0045] The recovered double-digestion products were ligated using Solution I ligase at 16℃ for 0.5–3 h to obtain the recombinant plasmid. The ligation system (10 μL) included: 1 μL vector fragment, 4 μL target gene, and 5 μL Solution I ligase. The recombinant plasmid was transformed into E. coli DH5α competent cells. After LB (Kana) selection, positive transformants were cultured, identified by PCR, and sequenced. If the sequencing results were correct, the pET28a(+)-HIC31 / HIC52 vector was successfully constructed.

[0046] 1.3 pET28a(+)-HIC31 / HIC52 transformed into Escherichia coli BL21(DE3) competent cells After culturing the correctly sequenced DH5α-pET28a(+)-HIC31 / HIC52 strain, plasmid extraction was performed. 1 μL of the pET28a(+)-HIC31 / HIC52 plasmid was mixed with thawed *E. coli* BL21(DE3) competent cells and incubated on ice for 30 min. The mixture was then heat-shocked at 42°C for 90 s and incubated on ice again for 5 min. 400 μL of antibiotic-free LB medium was added, and the mixture was incubated on a shaker at 37°C and 190 rpm for 40 min. 200 μL of the bacterial culture was evenly spread onto LB agar plates containing 50 μg / ml kanamycin (Kana). The plates were incubated upside down at 37°C overnight. The next day, transformed colonies were picked and inoculated. The plasmid was extracted from the overnight culture of the transformed colonies, and sequencing confirmed its accuracy, thus obtaining the recombinant fusion protein engineered strain BL21-pET28a(+)-HIC31 / HIC52.

[0047] 1.4 Induction of expression of BL21-pET28a(+)-HIC31 / HIC52 engineered bacteria Select a single colony of BL21-pET28a(+)-HIC31 / HIC52 with correct sequencing and inoculate it into 5 ml of LB (Kana) medium. Incubate overnight at 37°C with shaking at 180 rpm. Take 300 μL of the overnight bacterial culture at a 1% inoculation rate and inoculate it into an Erlenmeyer flask containing 30 ml of LB (Kana) medium. Incubate for 2.5 h until the initial OD600 reaches 0.6–0.8. Add IPTG inducer to a final concentration of 0.2 mM, and incubate at 37°C for 5 h.

[0048] After induction, the bacterial cell pellet was collected by centrifugation, and the supernatant was analyzed by 12% SDS-PAGE electrophoresis. The results are shown below. Figure 1 (M: Marker; 1. Supernatant of bacterial cell lysis induced by empty vector plasmid; 2. Supernatant of bacterial cell lysis induced by pET28a(+)-HIC31 / HIC52), After induction by BL21-pET28a(+)-HIC31 / HIC52, the supernatant obtained from bacterial cell lysis showed a specific band at around 28.8 KD, while the supernatant obtained from bacterial cell lysis of the BL21-pET28a(+) empty vector strain after induction did not show a specific band.

[0049] 1.5 Purification of HIC31 / HIC52 fusion protein The induced BL21-pET28a(+)-HIC31 / HIC52 cells were resuspended in 10× 20mM PB + 1M sodium chloride (pH 5.8) solution for at least 30 min. The cells were then autoclaved at 500 bar once and 1000 bar three times. The autoclaved bacterial suspension was balanced and centrifuged at 4℃, 8000 rpm for 30 min. The supernatant was collected, and 20mM PB (pH 5.8) was added to the supernatant to adjust the conductivity to 40±2 mS / cm. The suspension was filtered through a 0.45 μm membrane and precipitated using a Qianchun Bio SP 6 HP 5mL column and a Taidu Biopurifier. The baseline was stabilized by equilibration with solution A (20mM PB + 0.4M sodium chloride (pH 5.8)). Initial loading was 2 mL / min. After loading, the column was equilibrated again with solution A (20mM PB + 0.4M sodium chloride (pH 5.8) to ensure a stable baseline. 5.8) Pass the chromatography column to wash away unbound contaminating proteins until the UV baseline stabilizes. Then, pass the column through solution B (20 mM PB, 1.5 M sodium chloride, pH 5.8) with elution starting at 100% B / 10 CV. Collect the protein corresponding to the elution peak to obtain the HIC31 / HIC52 fusion protein stock solution. Analyze the purified protein using 12% SDS-PAGE electrophoresis. The results are shown below. Figure 2 (M: Marker; 1: Before loading HIC31 / HIC52; 2: Washing; 3: Elution) Purity is higher than 90%.

[0050] 1.6 Lyophilization of HIC31 / HIC52 fusion protein Table 1. Vacuum freeze-drying process of HIC31 / HIC52 fusion protein

[0051] Example 2: HIC31 / HIC52 fusion protein cytotoxicity assay 2.1 Experimental Methods Cytotoxicity was detected using the MTT assay. Human immortalized keratinocytes (HaCat) in the logarithmic growth phase were divided into groups of 2 × 10⁻⁶ cells. 4Cells were seeded at 100 μL / well in 96-well plates and cultured for 24 h in an incubator (37℃, 5% CO2). When the cell deposition rate in the 96-well plates reached 40%–60%, the following treatments were administered: ① Sample group: 100 μL of culture medium containing different concentrations (10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL, 0.156 mg / mL, 0.078 mg / mL) of HIC31 / HIC52 fusion protein was added to each well; ② Control group (NC): 100 μL of culture medium was added; ③ Blank group (BC): No cells were added, only 100 μL of culture medium was added; cultured for 24 h. The medium was discarded and replaced with 200 μL of culture medium containing MTT (0.5 mg / mL), and cultured for another 4 h. Add 100 μL / well of DMSO to the discarded solution, shake thoroughly, allow to develop color, and then measure the OD using a microplate reader. 490 Calculate the relative cell viability according to formula (1): Relative cell viability (%) = ×100% ………(1) Criteria for determining cytotoxicity: If the cell survival rate is greater than 70%, it is considered that there is no cytotoxic reaction; otherwise, it is considered that there is a cytotoxic reaction.

[0052] 2.2 Experimental Results Table 2 Results of cytotoxic MTT assay

[0053] The results of the cytotoxicity experiment are shown in Table 2, and the generated bar chart is shown in [Table 2]. Figure 3 When the HIC31 / HIC52 fusion protein was used in HaCat cells at a concentration ≤10.0 mg / mL for 24 h, the cell survival rate was greater than 70%, indicating that the fusion protein designed in this invention is not toxic to cells.

[0054] Example 3: Inhibition experiment of HIC31 / HIC52 fusion protein on melanin synthesis-related proteins. 3.1 Administration Human melanoma cells SK-MEL-28 in the logarithmic growth phase were divided into groups of 5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well into 6-well plates and incubated overnight in an incubator (37°C, 5% CO2). When the cell deposition rate in the 6-well plates reached 40%–50%, the cells were administered drugs in groups (0.156 mg / mL and 0.078 mg / mL), with 2 mL of drug administered to each well, and one well per group. After drug administration, the 6-well plates were incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0055] 3.2 Total RNA extraction from cells 3.2.1 Sample preparation.

[0056] Cell samples: Collect approximately 1 million cells, aspirate the culture medium, wash three times with PBS, add 300 μl of lysis buffer, gently pipette 5-10 times until the suspension dissolves and the solution becomes clear, then transfer to a clean centrifuge tube.

[0057] 3.2.2 Add an equal volume of binding solution to the lysis solution and gently invert to mix 3-5 times.

[0058] 3.2.3 Transfer the mixture (including the precipitate) to a new 1.5 ml centrifuge tube, add 20 µl of BeyoMag™ RNA magnetic bead suspension (be sure to mix well before use), mix gently, and incubate at room temperature for 3-5 minutes. Place the centrifuge tube in the magnetic field of the magnetic rack, and after the magnetic beads have completely aggregated, carefully aspirate any remaining liquid.

[0059] 3.2.4 Add 600µl of washing solution I, gently shake to disperse the magnetic beads, invert twice, and then place the centrifuge tube in the magnetic field of the magnetic rack. After the magnetic beads have completely gathered, try to remove as much residual liquid as possible.

[0060] 3.2.5 Add 600µl of washing solution II, gently shake to disperse the magnetic beads, invert twice, and then place the centrifuge tube in the magnetic field of the magnetic rack. After the magnetic beads have completely gathered, try to remove as much residual liquid as possible.

[0061] 3.2.6 Repeat step 5.2.5 once.

[0062] 3.2.7 Place the centrifuge tubes at room temperature for 5-10 minutes, or in a 37°C forced-air oven for 5 minutes, to ensure that any residual ethanol or other trace amounts of liquid completely evaporate.

[0063] 3.2.8 Add 50-100 μl of elution buffer, gently shake to suspend the magnetic beads in the solution, and incubate at room temperature for 3-5 minutes, shaking the centrifuge tube 1-2 times during this period. Place the centrifuge tube in a magnetic field, and after the magnetic beads have completely aggregated, carefully aspirate the solution into a new centrifuge tube and store at -20℃. The resulting solution is the extracted RNA.

[0064] The purity of the extracted RNA was determined using an ultra-micro spectrophotometer, OD. 260 / OD 280 The values ​​were all between 1.8 and 2.0, indicating that the next step of reverse transcription experiment could be carried out.

[0065] 3.3 Reverse transcription experiment 3.3.1 Refer to Table 3 to set up the reverse transcription reaction. Table 3. Preparation of Reverse Transcription Reaction System

[0066] *To 12μl means adding DEPC-treated Water to a final volume of 12μl.

[0067] 3.3.2 Mix gently, then centrifuge to precipitate the liquid.

[0068] 3.3.3 Incubate at 42℃ for 60 min.

[0069] 3.3.4 Incubate at 80℃ for 10 min.

[0070] 3.3.5 The reverse transcription product can be used directly for subsequent real-time quantitative PCR experiments, or it can be frozen at -20℃ for later use.

[0071] 3.4 Real-time quantitative PCR experiment The expression level of the gene associated with skin whitening, tyrosinase (TYR), was detected.

[0072] 3.4.1 Primers used for gene detection are shown in Table 4. Table 4 Primer Information for Quantitative Real-Time PCR

[0073] 3.4.2 Reaction System The PCR reaction system is shown in Table 5.

[0074] Table 5. Preparation of Real-Time Quantitative PCR Reaction System

[0075] 3.4.3 Standard Procedure The PCR reaction procedure is shown in Table 6.

[0076] Table 6 Real-time quantitative PCR reaction procedure

[0077] 3.4.4 Results Analysis Use 2 -△△CT The results were calculated using the following methods. T-tests were performed on the values ​​of the sample group and the NC group using software. A p-value < 0.05 was considered statistically significant compared to the SC group (denoted by *), and a p-value < 0.01 was considered highly statistically significant compared to the SC group (denoted by **).

[0078] The results of real-time PCR gene detection are shown in Table 7, and the generated bar chart is shown in Table 7. Figure 4 The results showed that the HIC31 / HIC52 fusion protein designed in this invention reduced the expression level of the h-TYR gene in human melanoma cells SK-MEL-28, indicating that the HIC31 / HIC52 fusion protein has a whitening effect.

[0079] Table 7 Results of Real-Time PCR Gene Detection

[0080] Example 4: Inhibition rate experiment of HIC31 / HIC52 fusion protein on tyrosinase activity 4.1 Experimental Methods In the activity assay system of 0.25 mol / L phosphate buffer (pH 6.8), the reaction solution was prepared according to Table 8: Table 8 Solution Preparation Table

[0081] Note: The sample was a 1 mg / mL HIC31 / HIC52 fusion protein solution dissolved in pure water; the tyrosinase activity was 100 U / mL.

[0082] After the C2 solution is prepared and shaken well, it is heated in a 37°C water bath for 10 minutes, with the wavelength adjusted to zero at 475nm.

[0083] After preparing the C1 solution, shake well and incubate in a 37°C water bath for 10 minutes. Then add 1 mL of tyrosinase, continue incubating in a water bath for another 10 minutes, and measure the absorbance value of C1.

[0084] Using the same method described above, the absorbance value of T1 was measured by zeroing at T2.

[0085] Calculate the inhibition rate T (%) of the sample on tyrosinase activity using the following formula: T (%) = (C1 - T1) / C1 × 100% 4.2 Experimental Results Experimental results show that the HIC31 / HIC52 fusion protein of the present invention inhibits tyrosinase activity by 87.6% at a concentration of 1 mg / mL, and has a strong whitening effect.

[0086] Example 5: Antioxidant experiment of HIC31 / HIC52 fusion protein 5.1 Solution Preparation ABTS solution: Weigh 200.0 mg of ABTS and 34.4 mg of potassium persulfate, dissolve in 50.0 mL of distilled water, shake well, and let stand at room temperature in the dark for 24 h to obtain the ABTS stock solution. Take an appropriate amount of the ABTS stock solution and dilute with 95% ethanol until the absorbance value is within 0.70 ± 0.02 (OD). 734 This solution is used as the ABTS assay solution and should be prepared fresh for immediate use.

[0087] HIC31 / HIC52 fusion protein solution: Weigh 20.0 mg of sample, dilute to 1 mL with distilled water, mix thoroughly to prepare a 20 mg / mL stock solution. Dilute the stock solution with distilled water to different concentrations to obtain sample solutions of different concentrations.

[0088] Glutathione solution: Weigh 10.0 mg of L-reduced glutathione, dilute to 1 mL with distilled water, mix thoroughly to prepare a 10 mg / mL stock solution. Dilute the stock solution with distilled water to different concentrations to obtain glutathione solutions of different concentrations.

[0089] 5.2 Experimental Procedure 1) Control group: Take two test tubes, numbered 1 and 2 respectively, and add reagents to each test tube according to the combination in Table 9. After mixing thoroughly, react at room temperature in the dark for 5 min, and measure the absorbance value at a wavelength of 734 nm using a UV spectrophotometer (zero calibration with sample solvent).

[0090] Table 9. ABTS Method Reagent Addition Amount

[0091] 2) HIC31 / HIC52 fusion protein group: The sample solution was used instead of the glutathione solution, and other operations were the same as the control group.

[0092] Data processing Calculate according to formula (2): P = ×100% …………………(2) In the formula: P — clearance rate; Ab —— (Test tube No. 2) Absorbance of the mixture of ABTS solution and sample solvent; As — (Test tube 1) Absorbance of the mixture of the test solution and ABTS solution; A linear equation (R² ≥ 0.9500) was established between the natural logarithm of the concentration of the test solution and the clearance rate (R² ≥ 0.9500) to calculate the half-maximal clearance (EC). 50 The antioxidant capacity AO value of the polypeptide sample is calculated according to formula (3).

[0093] AO= …………………(3) In the formula: AO – Antioxidant capacity; EC 50 (S)——Half-clearance of peptide sample, in milligrams per liter (mg / L). EC 50(R) – Half-maximal clearance of glutathione, expressed in milligrams per liter (mg / L). The calculation results are expressed as the arithmetic mean of parallel measurements, and are retained to three significant figures.

[0094] 5.3 Test Results ABTS was conducted using glutathione solutions of five different concentrations. + The free radical scavenging test, and the standard curve plotted are shown in [reference needed]. Figure 5 The calculation results are shown in Table 10. ABTS was carried out using six different concentrations of HIC31 / HIC52 fusion protein solutions. + The free radical scavenging test, and the standard curve plotted are shown in [reference needed]. Figure 6 The calculation results are shown in Table 11.

[0095] Table 10 Results of Glutathione ABTS Method

[0096] Table 11 Results of ABTS assay for HIC31 / HIC52 fusion protein

[0097] The test results showed that, according to the ABTS method, the half-maximal clearance (EC50) of glutathione was... 50 The half-maximal clearance (EC50) of the HIC31 / HIC52 fusion protein was 14.64 mg / L. 50 The antioxidant capacity of the HIC31 / HIC52 fusion protein is 2599.69 mg / L, and its AO value is 178; that is, the HIC31 / HIC52 fusion protein has antioxidant effects.

[0098] Example 6: Human skin patch test of HIC31 / HIC52 fusion protein 6.1 Experimental Methods Test substance: 10 mg / mL HIC31 / HIC52 fusion protein solution.

[0099] A total of 30 participants were selected according to the inclusion criteria. A qualified plaster applicator was used, and the test substance was placed inside the applicator at a volume of approximately 0.020 ml to 0.025 ml. The control well served as a blank control (without any substance). The plaster applicator containing the test substance was applied to the flexor side of the participant's forearm using non-irritating adhesive tape, and the applicator was gently pressed with the palm of the hand to ensure even application to the skin. This process was continued for 24 hours.

[0100] The test substance was removed after 24 hours. Skin reactions were observed and recorded at 0.5 hours, 24 hours, and 48 hours after patch removal, according to the skin reaction grading standards in the "Cosmetic Safety Technical Specifications" (2020 edition) (see Table 12). The results are shown in Table 13. No adverse reactions were observed in any of the 30 participants after 48 hours.

[0101] Table 12 Grading Standards for Skin Reactions in Closed Patch Tests

[0102] Table 13 Statistical Table of Results of Human Skin Occlusive Patch Experiment

[0103] Example 7: Determination of Skin Stratum Corneum Moisture Content of HIC31 / HIC52 Fusion Protein 7.1 Experimental Methods The prepared HIC31 / HIC52 fusion protein was formulated into an aqueous solution with a concentration of 1 mg / mL as the test sample. Thirty healthy women / men aged 25-50 years were recruited as test subjects. Subjects were divided into different test groups. Each group first cleansed their face with a designated cleanser sample and sat quietly for 30 minutes in a laboratory at a temperature of 21±1℃ and humidity of 50±10%RH. The stratum corneum moisture content of a 3cm×3cm area on the right upper cheek was measured using a Corneometer CM825 (before use). Then, the subject gently applied an appropriate amount of the test sample to the skin of the right upper cheek twice daily, morning and evening, for 14 and 28 consecutive days. The stratum corneum moisture content was measured again using a Corneometer CM825 after each application. A higher measured value indicates a higher stratum corneum moisture content.

[0104] 7.2 Experimental Results Statistical analysis was performed on the skin stratum corneum moisture content data measured at baseline (D0), 14 days (D14), and 28 days (D28), such as... Figure 7 As shown, the HIC31 / HIC52 fusion protein, when used in an aqueous solution at a concentration of 1 mg / mL for 14 and 28 days, can significantly increase the moisture content of the stratum corneum of the skin, indicating that the HIC31 / HIC52 fusion protein has a certain moisturizing effect.

[0105] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A pearl fusion protein with whitening effects, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID No:2; Alternatively, the fusion protein is a derived protein that has one or more amino acids substituted, deleted, or added to the amino acid sequence shown in SEQ ID No:2, and has the activity of the protein shown in SEQ ID No:

2.

2. A gene encoding the fusion protein of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No:

1.

4. A recombinant plasmid, characterized in that, The recombinant plasmid includes the gene described in claim 2 or 3.

5. An engineered bacterium, characterized in that, The engineered bacteria include the recombinant plasmid as described in claim 4.

6. A method for preparing a pearl fusion protein with whitening effects, characterized in that, Includes the following steps: S1. Based on the functional region sequences of HIC31 and HIC52, the matrix proteins of the Triangular Sail mussel, the properties of the pET28a(+) vector, and the host codon adaptability of Escherichia coli, the gene sequence and amino acid sequence of the fusion protein were designed and obtained using bioinformatics analysis software, and then the gene was synthesized. S2, based on the gene sequence of the fusion protein obtained in S1, construct the pET28a(+)-HIC31 / HIC52 vector; S3, the pET28a(+)-HIC31 / HIC52 vector was transformed into Escherichia coli competent cells to obtain engineered bacteria BL21-pET28a(+)-HIC31 / HIC52; S4. After inducing expression of the engineered bacteria BL21-pET28a(+)-HIC31 / HIC52, the bacterial cell precipitate was collected by centrifugation, the supernatant was obtained by lysis, and the fusion protein was obtained by purification.

7. The method according to claim 6, characterized in that, The method further includes S5: freeze-drying the fusion protein obtained in S4.

8. The method according to claim 6, characterized in that, The S2 of the method specifically includes the following steps: S21, the plasmid pET28a(+) and the plasmid PUC57-HIC31 / HIC52 were double-digested with NcoI and HindIII, respectively; S22. After the reaction was completed, the enzyme digestion system was subjected to electrophoresis on an agarose gel, and the double enzyme digestion products pET28a(+)(NcoI+HindIII) and HIC31 / HIC52(NcoI+HindIII) were recovered using a gel recovery kit. S23, the recovered double digestion product is ligated using Solution I ligase at 16°C for 0.5-3 hours; each 10 μL ligation system includes: 1 μL vector fragment, 4 μL target gene, and 5 μL Solution I ligase; S24. The recombinant plasmid system ligated from S23 was transformed into E. coli DH5α competent cells. After LB (Kana) resistance selection, positive transformants were selected and cultured. After PCR identification, sequencing was performed. If the sequencing results were correct, the pET28a(+)-HIC31 / HIC52 vector was successfully constructed. The pET28a(+)-HIC31 / HIC52 vector was extracted using a plasmid miniprep kit.

9. The method according to claim 6, characterized in that, The S3 of the method specifically includes the following steps: Mix 1 μL of pET28a(+)-HIC31 / HIC52 plasmid with thawed E. coli BL21(DE3) competent cells and incubate on ice for 30 min; then heat shock in a 42°C water bath for 90 s and incubate on ice again for 5 min. Add 400 μL of antibiotic-free LB medium and incubate at 37°C and 190 rpm for 40 min on a shaker. Spread 200 μL of the bacterial culture evenly onto an LB agar plate containing 50 μg / ml kanamycin. Incubate overnight at 37°C with the plate upside down. The next day, pick the transformed colonies for inoculation and culture. Extract the plasmid from the overnight culture of the transformed colonies. If the nucleotide sequencing is correct, the recombinant fusion protein engineered bacteria BL21-pET28a(+)-HIC31 / HIC52 is obtained.

10. The use of the protein according to claim 1 or the protein prepared by the method according to any one of claims 6-9, characterized in that, The application includes any of the following: Applications in reducing the expression level of tyrosinase genes or inhibiting tyrosinase activity; In ABTS + Applications in free radical scavenging or antioxidation; Applications in skin whitening or maintaining the moisture content of the stratum corneum; Applications in the preparation of cosmetics or medical aesthetic products.

Images